It is known that human tissue plasminogen activator (t-PA) possesses useful fibrinolytic activity in extremely efficiently activating fibrin-bound plasminogen, while it does not efficiently activate plasminogen in the circulating body fluid phase in contrast to ordinary thrombolytic agents, streptokinase (SK) and urokinase (UK). The amino acid sequence constituting the human t-PA and nucleotide sequence of cDNA coding for human t-PA are known [Pennica, D., et al., Nature, 301, 214-221 (1983)]. It is also known that human t-PA dissolves venous and arterial blood clots. In large scale clinical tests, it is reported that human t-PA given intravenously is effective for re-perfusion of obstructive coronary artery in the patient with acute myocardial infarction.
However, a defect in applying this human t-PA to the treatment of thrombotic disease is an extremely short half life of its enzyme activity in blood [Rijken, D.C., et al., Thromb Heamost., 54 (1), 61 (1985), Hubert, E. F., et al., Blood, 65, 539 (1985)]. Accordingly, when used for the treatment of thrombotic disease, human t-PA should be continuously administered intravenously in a high dose.
It is known that naturally occurring human t-PA takes a domain structure, from the N-terminal of the molecule, of the finger domain, the EGF (epidermal growth factor) domain, the two domains of kringle 1 and kringle 2 and the serine protease domain, based on its fully anticipated secondary structure. It is reported by Rijken et al. [Rijken, D.C., et al., Thromb. Haemost., 54 (1), 61 (1985)] that domains of human t-PA other than the serine protease domain would be responsible for the shortness of biological half life of human t-PA. It is also reported by Zonneveld et al. [Zonneveld, A.J.V., et al., Proc. Natl., Acad. Sci. U.S.A., 83, 4670 (1986)] that the finger domain, the EGF domain and the kringle 2 domain structure would take an important role for fibrin binding activity of naturally occurring human t-PA and for maintaining fibrin-dependent activation of t-PA. However, any concrete measure for maintaining fibrin binding activity possessed by naturally occurring human t-PA and desirable properties of fibrin-dependent activity and prolonging the biological half life are unknown.
In Published Unexamined Japanese Patent Application Laid Open No. 48378/1987, there is described t-PA obtained by deletion of 87th to 175th amino acids of naturally occurring human t-PA in which kringle 1 is deleted. This t-PA is characterized by further inducing site mutation in the EGF region. The Japanese Patent Application discloses that the modified t-PA has a binding ability to fibrin and interaction with a tissue plasminogen activator inhibitor is reduced.
In EP No. 241208, there is described t-PA obtained by deletion of 92nd to 179th amino acids of naturally occurring human t-PA in which kringle 1 is deleted. It is simply mentioned that this t-PA has a fibrinolytic activity.
Furthermore, EP No. 231624 discloses modified 10 t-PA showing a prolonged half life. The modified t-PA having F-EGF-K2-A as one of its sequence is deleted of kringle 1 but any specific process for its production is not shown. It is understood that the modified t-PA in the present invention is different from naturally occurring one in the amino acid sequence in the inter-domain region, in light of the specifically recited process.
As a result of extensive investigations, the present inventors have produced an improved t-PA which contains the finger domain, the EGF domain, the kringle 2 domain and the serine protease domain but the first kringle 1 domain is deleted at a specific amino acid site and further produced an improved t-PA which has caused site-specificmutation at the kringle 2 domain-serine protease linking site and, have succeeded in obtaining improved t-PA having excellent stability to heat and acids, having markedly prolonged biological half life and also having an antiinflammatory activity, while unexpectedly maintaining desirable properties of naturally occurring human t-PA.